1. Field of the Invention
The present invention relates to the field of biological cell-to-cell communication and electrochemical signalling between biological cells. In particular, the present invention provides a process for identifying substances that modulate the activity of hyperpolarization-activated cation channels, and the use of this process.
2. Description of the Relevant Art
Some genes of murine and human hyperpolarization-activated cation channels are already known. Examples include muHCN2(muHAC1) (Ludwig et al. (1998)), huHCN4 (Ludwig et al. (1999)), huHCN2 (Vaccari, T. et al. (1999) Biochim. Biophys. Acta 1446(3): 419-425), and those disclosed in WO 99/32615 and WO 99/42574. See, also, Tables 1-6 herein.
Ludwig et al. (1998) have shown that muHCN2 can be transfected transiently in HEK293 cells, and that the corresponding channel in the transfected cells can be examined easily by electrophysiological methods (patch-clamp studies). The electrophysiological properties of the cloned channel correspond to the lf or lh current described in pacemaker cells, which had hitherto not been known on a molecular level (Ludwig et al. (1998), Biel et al. (1999)). The channel activates when the holding potential is changed toward hyperpolarization (potential at about B100 to B160 mV). However, the patch-clamp technique cannot be automated and is not suitable for high-throughput screening (HTS).
Using suitable dyes, ion currents can be measured in an FLIPR (fluorescence imaging plate reader; Molecular Devices, Sunnyvale Calif., USA). Influx or efflux of ions leads to changes in the membrane potential, which can be measured in high-throughput screening in an FLIPR using suitable fluorescent dyes. However, in contrast to the patch-clamp method, it is not possible to generate voltage changes in the FLIPR. Voltage changes are, however, an essential prerequisite for the activation of hyperpolarization-activated cation channels.